Ⅰ型启动子介导的植物RNA病毒载体表达系统的研究

As a novel expression system, plant virus RNA-based vectors allow for the rapid, high level, transient expression of proteins in whole plants. Many different types of plant viruses have been converted into vectors for the production of recombinant proteins or peptides and they all used the type II promoters. However, under natural conditions, plant RNA viruses replicate in the cytoplasm and are not adapted to the nuclear splicing machinery, which recognizes and removes cryptic introns from viral RNA. As a result, pre-mRNA transcripts made in the nucleus from viral constructs may not be recognized and processed properly and the formation of active replicons from the primary nuclear transcript is inefficient. RNA Pol I is a cellular enzyme that is abundantly expressed in cells and transcribes rRNA precursor lacking a 5′ cap, a 3′ poly (A) tail and introns. Thus, viral RNA synthesized in cells transfected with Pol I-driven plasmids containing viral genomic cDNA has precise sequences. This may increase the expression level and biological safety of the plant RNA-based expression system. In addition to, Nicotiana benthaminana is a kind of model organism which widely used in plant bioreactor and plant pathology. For these reasons, we plan to clone the Nicotiana benthaminana Pol I promoter sequence and analyses its transcription initiation site. And then the TBSV will be used to construct the infectious cDNA vectors under control of the Nicotiana benthaminana rRNA promoter. Based on those constructed vectors, we will verify the cis-elements and its function in the Nicotiana benthaminana Pol I promoter sequence and exploit if Pol I-directed transcription require nuclear splicing. After that, a RNA polymerase I-mediated plant virus RNA-based expression system will be established. The biological safety and utilities of this Pol I-driven plant RNA-based expression system also be evaluated simultaneously. The related results from this study will be beneficial for the further research of both Pol I promoter and Pol I-based plant virus expression vectors. Moreover, this new transient expression system will be a useful tool for expressing recombinant proteins in plants for either research or production purposes.

植物RNA病毒载体表达系统是一种极具潜力的新型外源基因表达平台。当前植物RNA病毒载体均使用Ⅱ型启动子，与Ⅱ型启动子伴生的转录后加工（加帽、加尾和内含子剪接等）会影响重组病毒基因组RNA的完整性，导致载体表达效率下降、外源基因丢失和产物不均一等问题。Ⅰ型启动子转录活性高、转录产物无需类似加工且具有种属间特异性，应用于病毒表达载体即利于保持转录后重组病毒基因组的完整性，也利于提高系统的生物安全性。鉴于此，本项目在克隆本氏烟Ⅰ型启动子的基础上，以番茄丛矮病毒为实验对象，以GFP为报道基因，构建本氏烟Ⅰ型启动子介导转录的病毒表达载体。通过相关研究，探明启动子中相关顺式调控元件及其功能；明确转录后RNA加工对病毒表达系统的影响；建立Ⅰ型启动子介导转录的病毒表达系统并评价系统在提高生物安全性和实用性上的潜力。研究对深入理解植物病毒表达系统和指导新类型病毒表达载体的构建方面均具有理论意义和借鉴价值。

植物RNA病毒载体表达系统是一种极具潜力的新型外源基因表达平台。使用Ⅱ型启动子的植物RNA病毒表达载体，转录后加工会影响重组病毒基因组RNA的完整性，导致载体表达效率下降、外源基因丢失和产物不均一等共性问题。Ⅰ型启动子具有种属间特异性且其转录产物无需类似的转录后加工环节，应用于病毒表达载体不仅可以规避上述问题，而且还有提高生物安全性的可能性。目前，I型启动子被广泛应用于动物病毒载体的研究中，但在植物中却鲜有报道。鉴于此，本项目在克隆本氏烟Ⅰ型启动子的基础上，以番茄丛矮病毒（TBSV）为实验对象，以GFP为报道基因，构建本氏烟Ⅰ型启动子病毒表达载体。通过相关研究，探索将Ⅰ型启动子应用于植物病毒表达载体中的可行性和应用潜能。研究发现，（1）本氏烟Ⅰ型启动子转录起始位点位于高度保守的核心序列中的第3位A处；（2）启动子长度对转录效率无显著影响，77 bp启动子序列就能够起始病毒基因组RNA的转录，启动子结构中不存在类似于动物I型启动子的上游控制元件UCE；（3）转录起始位点下游延伸序列对启动子转录强度具有显著的正调控作用，能够提高病毒载体表达水平2倍左右；（4）来源于35S启动子的ELS序列能够增强启动子转录频率和提高病毒载体表达水平3-5倍；（5）优化后的I型启动子具有与35S启动子相同的转录强度；（6）本氏烟I型启动子具有低水平地转录非专一性，Pol II对I型启动子的非特异性识别是现象产生的可能原因；（7）对I型启动子核心序列进行单碱基突变，可以在不影响启动子转录效率的前提下提高其转录专一性；（8）I型终止子的存在与否不影响病毒载体的表达效率，推测TBSV病毒对其3’末端的高水平修复是产生问题的可能原因；（9）I型启动子在一定程度上缩小了病毒载体的寄主范围，具有提高病毒载体生物安全性的潜力；（10）Ⅰ型启动子TBSV病毒载体可以高效地表达小片段多肽。相关结果不仅有助于更新人们对I型启动子结构特点和转录特点的认识，也为植物源I型启动子结构研究提供了简便的方法。同时，研究结果也为植物病毒载体表达系统领域中的一些共性问题的解决提供了新的思路。